Jerome J. Perry

Bacillus thermokovorans, sp. nov., a Species of Obligately Thermophilic Hydrocarbon Utilizing Endospore-forming Bacteria

Summary Ten strains of obligately thermophilic bacteria able to utilize w-alkanes as growth substrate were isolated from a variety of environmental sources. All are aerobic, endospore-forming rods with the vegetative cells giving a generally negative Gram reaction. The organisms grow at a temperature from 42–75°C with optimum growth between 55–65°C. Optimum pH for growth is 6.2–7.5. The mol % G+C values range from 52–58. All strains lack pigmentation and none are motile. The organisms were further characterized on the basis of biochemical and nutritional properties and electrophoretic mobility of catalase, superoxide dismutase and esterase. DNA-DNA hybridization was performed to determine the taxonomic relatedness of the 10 strains. Results from relative reassociation reactions indicate 3 homology groups (60–100% relative reassociation). There was significant relatedness to Bacillus stearotbermopbilus (46–67% relative reassociation). No relationship was observed between these 10 strains and any mesophilic species of Bacillus tested or to the obligate thermophiles Thermus aquaticus, Thermomicrobium roseum and Thermoleopbilum album . These results were further substantiated by determining the thermal stabilities of reassociated hetero-duplexes. The ΔTm (e) values for heterologous duplexes formed with DNA from the thermophilic bacteria and B. stearotbermopbilus strains ranged between 0–9°C while values obtained with mesophilic and thermophilic bacilli had an average ΔTm (e) = 17°C ± 2°C. The designation Bacillus thermoleovorans is proposed to describe these strains, and the type strain is LEH-1 (ATCC 43513).

Microbial Degradation of Cycloparaffinic Hydrocarbons via Co-metabolism and Commensalism

SUMMARY Mineralization of a cycloparaffinic hydrocarbon in soil has been demonstrated. Addition of [U-14C]cyclohexane to marine mud, followed by incubation at 26 °C, resulted in the evolution of 14CO2. All attempts in our laboratory to isolate organisms from soil which utilize cycloparaffins as a sole source of carbon and energy have proved unsuccessful. However, several hydrocarbon-utilizing bacteria oxygenated cycloparaffins to ketone derivatives that serve as the energy and carbon source for numerous other soil micro-organisms. The concerted attack of a mixed microbial population on cyclohexane has been demonstrated, suggesting that both co-metabolism and commensalism are associated with microbial degradation of cycloparaffinic hydrocarbons.

Thermoleophilum album gen. nov. and sp. nov., a bacterium obligate for thermophily and n-alkane substrates

Several bacterial strains that are obligate for both thermophily and hydrocarbon utilization have been isolated from a number of thermal and non-thermal environments. Mud and water samples obtained from geographic sites across the United States were subjected to enrichment procedures at 60° C with n-heptadecane as sole growth substrate. Organisms forming very small white colonies on agar surfaces were often evident on primary enrichment. These bacteria were Gram negative, aerobic, small, and rodshaped. They lacked pigmentation, motility, and the ability to form endospores. Growth occurred in the temperature range from 45° C to 70° C with the optimum around 60° C and at a pH near neutrality. Only n-alkanes from 13 to 20 carbons in length were utilized by these organisms as growth substrate. The mol% guanine plus cytosine values for these strains were between 68 and 70%. The physiological and morphological characteristics of these organisms are distinctly different from any previously described thermophilic microbes. It is proposed that they be placed in a new genus, Thermoleophilum gen. nov. with the type species being Thermoleophilum album gen. nov., sp. nov. The type strain in ATCC 35263.

Catabolism of 2,4,6-trinitrotoluene byMycobacterium vaccae

Mycobacterium vaccae strain JOB-5 cometabolized 2,4,6-trinitrotoluene (TNT) in the presence of propane as a carbon and energy source. Two novel oxidized metabolites, as well as several known reduced products, were generated during catabolism of TNT byM. vaccae. During the cometabolic process, there was transient production of a brown chromophore. This compound was identified as 4-amino-2,6-dinitrobenzoic acid. WhenM. vaccae was incubated with [14CTNT and propane, 50% of the added radiolabel was incorporated into the cellular lipid fraction. These results suggest that ring cleavage occurred prior to the incorporation of radiolabelled carbon into phosphatidyl-l-serine, phosphatidylethanolamine, cardiolipin, and other polar lipids.

Co-metabolism as a factor in microbial degradation of cycloparaffinic hydrocarbons

SummaryAll attempts to isolate microoganisms from soil that utilize unsubstituted cycloparaffinic hydrocarbons, e.g. cyclohexane, as sole source of carbon and energy have been unsuccessful. However, cyclohexane was degraded in fertile soil as measured by release of 14C-carbon dioxide on addition of UL-14C-cyclohexane. Hydrocarbon utilizing organisms isolated from the soil grew rapidly on cycloalkanones. Several cultures, after growth on propane, could oxidize cycloparaffins to the homologous cycloalkanone. These results suggest that degradation of cycloalkanes in nature may be via co-metabolism.

Propane Utilization by Microorganisms

Publisher Summary Propane is of widespread occurrence in petroleum deposits and represents 1–2% of natural gas. It is of considerable advantage as a raw material in the fermentation industry as it is low in cost, yields a weight increase on conversion proportional to the amount of oxygen incorporated, and is relatively insoluble in water, so that the unused gaseous substrate could readily be recycled in continuous fermentations. Some of the strains that utilize propane include Mycobacterium rhodochrous strains OFS, Mycobacterium convolutum strain R-22, and M. rhodochrous strain OC2A. The yield from propane and ethane, on a gram per gram substrate basis, is quite high and in line with results for methane-utilizing organisms. Studies show that considerably greater growth can be attained with a continuous flow of propane or butane into the system. Propane has been reported to effectively inhibit bacterial spore germination. Propylene is readily converted to acrylic acid by propane-utilizing bacteria and acrylic acid is an effective inhibitor of fatty acid synthesis.

Purification and characterization of the secondary alcohol dehydrogenase from propane-utilizing Mycobacterium vaccae strain JOB-5.

Mycobacterium vaccae strain JOB-5 cultured in the presence of propane contained an inducible secondary alcohol dehydrogenase. The enzyme was purified 198-fold using DEAE-cellulose, omega-aminopentyl agarose and NAD-agarose chromatography. The Mr of the enzyme was approximately 136000, with subunits of Mr 37000. The pH optimum for the reaction oxidizing propan-2-ol to propanone was 10-10.5 while the optimum for the reverse reaction was 7.5-8.5. The isoelectric point was 4.9. NAD but not NADP could serve as electron acceptor. The apparent Km values for propan-2-ol and NAD were 4.9 X 10(-5)M and 2.8 X 10(-4)M, respectively. The enzyme was inhibited by thiol reagents and metal chelators. It appears to play an essential role in the metabolism of propane by this bacterium.

Effect of environmental pollutants and their metabolites on a soil mycobacterium

The relative toxicity of seven major ground-water pollutants (benzene, chlorobenzene, propylbenzene, ethylbenzene, trichloroethylene, toluene, and styrene) and their metabolites to a soil mycobacterium (Mycobacterium vaccae strain JOB-5) that can catabolize all of these pollutants was determined. The metabolites of chlorobenzene, styrene and trichloroethylene degradation (4-chlorophenol, styrene oxide, and 2,2,2-trichloroethanol, respectively) were less toxic to M. vaccae than was their parent compound. The pollutants propylbenzene, ethylbenzene and benzene were less toxic than their metabolites (4-propylphenol, 4-ethylphenol, and phenol). Metabolites were also examined for their ability to interfere with the biodegradation of selected groundwater pollutants. The metabolites of ethylbenzene, propylbenzene and chlorobenzene biotransformation by M. vaccae were found to adversely affect biodegradation by M. vaccae. Toluene degradation by M. vaccae was inhibited by 4-chlorophenol, 4-ethylphenol and 4-propylphenol at 0.2 mm, 0.4 mm, and 0.4 mm, respectively.

Changes in mutagenicity during crude oil degradation by fungi.

Two fungal strains, Cunninghamella elegans and Penicillium zonatum, that grow with crude oil as a sole carbon source were exposed to three crude oils that exhibit a range of mutagenic activity. At regular time intervals following fungal incubation with the various crude oils, extracts were tested for the presence of mutagenic activity using the spiral Salmonella assay. When the most mutagenic of the oils, Pennsylvania crude oil, was degraded by C. elegans or by P. zonatum, its mutagenicity was significantly reduced; corresponding uninoculated (weathered) controls of Pennsylvania crude remained mutagenic. West Texas Sour crude oil, a moderately mutagenic oil, exhibited little change in mutagenicity when incubated with either C. elegans or P. zonatum. Swanson River Field crude oil from Cook Inlet, Alaska is a slightly mutagenic oil that became more mutagenic when incubated with C. elegans; weathered controls of this oil showed little change in mutagenicity. Mycelial mat weights measured during growth on crude oils increased corresponding to the biodegradation of about 25% of the crude oil.

Paraquat toxicity and effect of hydrogen peroxide on thermophilic bacteria.

Paraquat (PQ++) increased cyanide-resistant univalent respiration in cell suspensions of five strains of obligately thermophilic bacteria. PQ++ was reduced by an NADH: or NADPH:paraquat diaphorase and selectivity for NADH, NADPH, or both electron donors varied among the thermophiles. Superoxide anion production that was dependent on the presence of PQ++ was shown by following the superoxide dismutase-inhibitable reduction of cytochrome c. In addition, the PQ++-dependent formation of hydrogen peroxide from superoxide anion was evident in two of the thermophilic strains. Catalase synthesis was induced by adding hydrogen peroxide to the growth medium of the thermophiles. The induction of catalase to eliminate hydrogen peroxide appears to be an important response of these thermophilic bacteria to oxygen toxicity.